Bacterial biofilms exist in natural, medical, and engineering environments. The biofilms offer a selective advantage to a microorganism to ensure its survival, or allow it a certain amount of time to exist in a dormant state until suitable growth conditions arise. Unfortunately, this selective advantage poses serious threats to animal health, especially human health.
Chronic infections involving biofilms are serious medical problems throughout the world. For example, biofilms are involved in 65% of human bacterial infections. Biofilms are involved in prostatitis, biliary tract infections, urinary tract infections, cystitis, lung infections, sinus infections, ear infections, acne, rosacea, dental caries, periodontitis, nosocomial infections, open wounds, and chronic wounds.
Compounds that modify biofilm formation would have a substantial medical impact by treating many chronic infections, reducing catheter- and medical device-related infections, and treating lung and ear infections. The potential market for biofilm inhibitors could be enormous given the sheer number of cases in which biofilms contribute to the medical problems. The inhibitors may be used to cure, treat, or prevent a variety of conditions, such as, but are not limited to, arterial damage, gastritis, urinary tract infections, pyelonephritis, cystitis, otitis media, otitis externa, leprosy, tuberculosis, benign prostatic hyperplasia, chronic prostatitis, chronic lung infections of humans with cystic fibrosis, osteomyelitis, bloodstream infections, skin infections, open or chronic wound infections, cirrhosis, and any other acute or chronic infection that involves or possesses a biofilm.
In the United States, the market for antibiotics is greater than $8.5 billion. After cardiovascular therapeutics, the sales of antibiotics are the second largest drug market in the United States. The antibiotic market is fueled by the continued increase in resistance to conventional antibiotics. Approximately 70% of bacteria found in hospitals resist at least one of the most commonly prescribed antibiotics. Because biofilms appear to reduce or prevent the efficacy of antibiotics, co-administration of biofilm inhibitors could significantly boost the antibiotic market.
Using the protection of biofilms, microbes can resist antibiotics at a concentration ranging from 1 to 1.5 thousand times higher than the amount used in conventional antibiotic therapy. During an infection, bacteria surrounded by biofilms are rarely resolved by the immune defense mechanisms of the host. It has been proposed that in a chronic infection, a biofilm gives bacteria a selective advantage by reducing the penetration of an antibiotic into the depths of the tissue needed to completely eradicate the bacteria's existence (Costerton J W et al., Science. 1999 May 21; 284(5418):1318-22).
Traditionally, antibiotics are discovered using the susceptibility test methods established by the National Committee for Clinical Laboratory Standards (NCCLS). The methods identify compounds that specifically affect growth or death of bacteria. These methods involve inoculation of a bacterial species into a suitable growth medium, followed by the addition of a test compound, and then plot of the bacterial growth over a time period post-incubation. Unfortunately these antibiotics derived from the NCCLS methods would not be effective therapeutics against chronic infections involving biofilms because the methods do not test compounds against bacteria in a preformed biofilm. Consistently, numerous publications have reported a difference in gene transcription in bacteria living in biofilms from bacteria in suspension, which further explains the failure of conventional antibiotics to eradicate biofilm infections (Sauer, K. et al. J. Bacteriol. 2001, 183:6579-6589).
Biofilm inhibitors can provide an alternative treatment approach for certain infections. Biofilm inhibitors, on the other hand, act on the biological mechanisms that provide bacteria protection from antibiotics and from a host's immune system. Biofilm inhibitors may be used to “clear the way” for the antibiotics to penetrate the affected cells and eradicate the infection. Traditionally, treatment of nosocomial infections requires an administration of a combination of products, such as amoxicillin/clavulanate and quinupristin/dalfopristin, or an administration of two antibiotics simultaneously. In one study of urinary catheters, rifampin was unable to eradicate methicillin-resistant Staphylococcus aureus in a biofilm but was effective against planktonic, or suspended cells (Jones, S. M., et. al., “Effect of vancomycin and rifampicin on methicillin-resistant Staphylococcus aureus biofilms”, Lancet 357:40-41, 2001).
Bacteria have no known resistance to biofilm inhibitors. Biofilm inhibitors are not likely to trigger growth-resistance mechanisms or affect the growth of the normal human flora. Thus, biofilm inhibitors could potentially extend the product life of antibiotics.
Biofilm inhibitors can also be employed for the treatment of acne. Acne vulgaris is the most common cutaneous disorder. Propionibacterium acnes, is the predominant microorganism present in acne. The bacteria reside in biofilms. The bacteria's existence in a biofilm matrix provides them with a protective, physical barrier that limits the effectiveness of antimicrobial agents (Burkhart, C. N. et. al., “Microbiology's principle of biofilms as a major factor in the pathogenesis of acne vulgaris”, International J. of Dermatology. 42:925-927, 2003). Biofilm inhibitors may be used to effectively prevent, control, reduce, or eradicate P. acnes biofilms in acne.
Plaque biofilms contribute to cavities and periodontitis. Plaque biofilms accumulate due to bacterial colonization of Streptococci spp., such as S. mutans, S. sobrinas, S. gordonii, and Porphyromonas gingivalis, and Actinomyces spp (Demuth, D. et al. Discrete Protein Determinant Directs the Species-Species Adherence of Porphyromonas gingivalis to Oral Streptococci, Infection and Immunity, 2001, 69(9) p 5736-5741; Xie, H., et al. Intergeneric Communication in Dental Plaque Biofilms. J. Bacteriol. 2000, 182(24), p 7067-7069). The primary colonizing bacteria of plaque accumulation are Streptococci spp., while P. gingivalis are a leading cause of periodontitis (Demuth, D. et al. Discrete Protein Determinant Directs the Species-Species Adherence of Porphyromonas gingivalis to Oral Streptococci, Infection and Immunity, 2001, 69(9) p 5736-5741). Biofilm inhibitors can be employed to prevent microorganisms from adhering to surfaces that may be porous, soft, hard, semi-soft, semi-hard, regenerating, or non-regenerating. These surfaces may be teeth, polyurethane material of central venous catheters, or metal, alloy, or polymeric surfaces of medical devices, or regenerating proteins of cellular membranes of mammals. These inhibitors can be coated on or impregnated into these surfaces at a concentration sufficient to control, reduce, or eradicate the microorganisms adherence to these surfaces.
Chronic wound infection represents another illness that is difficult to eradicate. Examples of the most common types of chronic wounds are diabetic foot ulcers, venous leg ulcers, arterial leg ulcers, and pressure ulcers. Diabetic foot ulcers appear to be the most prevalent. These wounds are typically colonized by multiple species of bacteria including Staphylococcus spp., Streptococcus spp., Pseudomonas spp. and Gram-negative bacilli (Lipsky, B. Medical Treatment of Diabetic Foot Infections. Clin. Infect. Dis. 2004, 39, p.S104-14).
Based on clinical evidence, microorganisms cause or contribute to chronic wound infections. Only recently have biofilms been implicated in these infections (Harrison-Balestra, C. et al. A Wound-isolated Pseudomonas aeruginosa Grow a Biofilm In Vitro Within 10 Hours and Is Visualized by Light Microscopy, Dermatol Surg 2003, 29; 631-635; Edwards, R. et al. Bacteria and wound healing. Curr Opin Infect Dis, 2004, 17; 91-96). Approximately 140,000 amputations occur each year in the United States due to chronic wound infections that could not be treated with conventional antibiotics. Unfortunately, treating these infections with high doses of antibiotics over long periods of time contributes to the development of antibiotic resistance (Howell-Jones, R. S., et al. A review of the microbiology, antibiotic usage and resistance in chronic skin wounds. J. Antimicrob. Ther. Jan. 2005). Biofilm inhibitors in a combination therapy with antibiotics may provide an effective alternative to the treatment of chronic wounds.
Recent publications describe the cycles of the pathogenesis of numerous species of bacteria involving biofilms. For example, Escherichia coli, which causes recurrent urinary tract infections, undergo a cycle of binding to and then invading a host's bladder epithelial cells. The E. coli form a biofilm intracellularly, modify its morphology, and then burst out of the host cells to repeat the cycle of pathogenesis (Justice, S. et al. Differentiation and development pathways of uropathogenic Escherichia coli in urinary tract pathogenesis, PNAS 2004, 101(5): 1333-1338). The authors suggest that this repetitive cycle of pathogenesis of E. coli may explain the recurrence of the infection.
In 1997, Finlay, B. et al. reported that numerous bacteria, including Staphylococci, Streptococci, Bordetella pertussis, Neisseria spp., Helicobactor pylori, and Yersinia spp., adhere to mammalian cells during their pathogenesis. The authors hypothesized that the adherence would lead to an invasion of the host cell. Later publications confirm this hypothesis (Cossart, P. Science, 2004, 304; 242-248; see additional references infra). Other publications presented similar hypotheses to Mulvey, M. et al. (Mulvey, M. et al. “Induction and Evasion of Host Defenses by Type 1-Piliated Uropathogenic E. coli” Science 1998, 282 p. 1494-1497). In particular, Mulvey, M. et al. stated invasion of E. coli into epithelial cells provide protection from the host's immune response to allow a build up of a large bacterial population.
Cellular invasion and biofilm formation appear to be integral to the pathogenesis of most, if not all bacteria. Pseudomonas aeruginosa have been shown to invade epithelial cells during lung infections (Leroy-Dudal, J. et al. Microbes and Infection, 2004, 6, p. 875-881). P. aeruginosa are the principal infectious organisms found in the lungs of cystic fibrosis patients, and the bacteria exist within a biofilm. Antibiotics like tobramcyin, and other current antibacterial compounds, do not provide effective treatment against biofilms of chronic infections, perhaps because antibiotic therapy fails to eradicate the biofilm.
The pathogenesis of cellular invasion and biofilm formation gram-negative bacteria follow conserved mechanisms. For example, Haemophilus influenzae invade epithelial cells and form biofilms (Hardy, G. et al., Methods Mol. Med., 2003, 71; 1-18; Greiner, L. et al., Infection and Immunity, 2004, 72(7); 4249-4260). Burkholderia spp. invade epithelial cells and form biofilm (Utaisincharoen, P. et al., Microb Pathog. 2005, 38(2-3); 107-112; Schwab, U. et al. Infection and Immunity, 2003, 71(11); 6607-6609). Klebsiella pneumoniae invade epithelial cells and form biofilm (Cortes, G et al. Infection and Immunity. 2002, 70(3); 1075-1080; Lavender, H. et al., Infection and Immunity. 2004, 72(8); 4888-4890). Salmonella spp. invade epithelial cells and form biofilms (Cossart, P. Science, 2004, 304; 242-248; Boddicker, J. et al., Mol. Microbiol. 2002, 45(5); 1255-1265). Yersinia pestis invade epithelial cells and form biofilms (Cossart, P. Science, 2004, 304; 242-248; Jarrett, C. et al. J. Infect. Dis., 2004, 190; 783-792). Neisseria gonorrhea invade epithelial cells and form biofilms (Edwards, J. et al., Cellular Micro., 2002, 4(9); 585-598; Greiner, L. et al., Infection and Immunity. 2004, 73(4); 1964-1970). Burkholderia sp. are another important class of gram-negative bacterial pathogens. Chlamydia sp., including Chlamydia pneumoniae is an intracellular, Gram-negative pathogen implicated in respiratory infections and chronic diseases such as atherosclerosis and Alzheimer's disease (Little, C. S. et al., Infection and Immunity. 2005, 73(3); 1723-34).
These Gram-negative bacteria cause lung, ear, and sinus infections, gonorrhoeae, plague, diarrhea, typhoid fever, and other infectious diseases. E. coli and P. aeruginosa are two of the most widely studied Gram-negative pathogens. Researchers believe that the pathogenesis of these bacteria involves invasion of host cells and formation of biofilms. These models have enabled those skilled in the art to understand the pathogenesis of other Gram-negative bacteria.
Gram-positive bacteria also share conserved mechanisms of bacterial pathogenesis involving cellular invasion and biofilm formation. Staphylococcus aureus invade epithelial cells and form biofilms (Menzies, B. et al., Curr Opin Infect Dis, 2003, 16; 225-229; Ando, E. et al., Acta Med Okayama, 2004, 58(4); 207-14). Streptococcus pyogenes invade epithelial cells and form biofilms (Cywes, C. et al., Nature, 2001, 414; 648-652; Conley, J. et al., J. Clin. Micro., 2003, 41(9); 4043-4048).
U.S. Pat. No. 4,606,911 (referred to as the '911 patent hereafter) describes compounds that selectively inhibit the growth and anti-adherence activities of Gram-positive mouth bacteria Streptococcus mutans but do not effect other bacteria. This patent discloses the use of oleanolic and ursolic acid as inhibiting the growth of S. mutans and promoting anti-adherence activities. The patent also lists compositions for oral care products in the claims. However, the patent clearly states the benefit of ursolic acid and related compounds is that they do not affect oral microorganisms other than S. mutans. Growth inhibition data presented in this patent indicated that ursolic acid completely inhibited S. mutans and S. salivaris (both gram-positive Streptococcal bacteria) yet failed to inhibit the gram-positive bacterium S. aureus (gram-positive) or the gram negative bacteria E. coli and P. aeruginosa. Oleanolic acid displayed incomplete inhibition of S. mutans and S. salivaris (both gram-positive bacteria) yet failed to inhibit the gram-positive bacterium S. aureus or the gram negative bacteria E. coli and P. aeruginosa. The '911 patent thus teaches that these compounds are useful for treating tooth decay by specifically inhibiting S. mutans growth and adherence. Consequently, the '911 patent neither demonstrates nor suggests that ursolic acid and oleanolic acid or the derivatives described herein prevent, inhibit, or reduce the in vitro or in vivo formation of biofilms. Furthermore, the '911 patent neither demonstrates nor suggests that ursolic acid and oleanolic acid can prevent or treat bacterial infections caused by microorganisms other than S. mutans. Moreover, the '911 patent does not teach or suggest use of ursolic acid and oleanolic acid in oral care products in combination with an antimicrobial agent or antibiotic. Finally, the '911 patent only teaches the use of pentacyclic acid triterpene compounds with hydrogen at position C-2 and hydroxyl at C-3 for inhibition of S. mutans and S. salivaris. As demonstrated in the examples, the compounds of this instant invention may be used in combination with antibiotics to treat chronic infections like plaque.
Honda, T.; et al., in “Design and synthesis of 2-Cyano-3,12-Dioxoolean-1,9-dien-28-oic acid, a novel and highly active inhibitor of nitric oxide production in mouse macrophages.” Bioorg. Medic. Chem. Lett., 1998, 8, 2711-2714, describe various oleanolic and ursolic acid derivatives including 3-hydroxy, 3-chloro-, and 2-chloro. However, this disclosure of oleanolic and ursolic acid derivatives was primarily concerned with discovery of compounds capable of inhibiting Interferon-γ induced nitric oxide production in mouse macrophages. Furthermore, the bulk of this disclosure focused on various enone-derivatives of the C-3 position of the ursane or oleanane scaffold. Finally, Honda et al neither demonstrates nor suggests that ursolic acid and oleanolic acid derivatives can prevent, inhibit, or reduce biofilm formation or bacterial infections caused by microorganisms.
Accordingly, for the reasons discussed above and others, there exists an unmet need for compounds that serve as biofilm inhibitors and/or that would be useful for preventing, reducing, or inhibiting bacterial infections.